There are a number of different types of vesicles released by cells, which are known as extracellular vesicles (EV). It is generally accepted that there are 3 main types of EVs which include apoptotic bodies (50-5,000 nm), microvesicles (100-1000 nm), and exosomes (40-150 nm). Apoptotic bodies are shed from dying cells whereas microvesicles are shed from the plasma membrane of viable cells. Exosomes are of endocrytic origin and are formed intracellularly by inward budding of the membrane of endocytic compartments, which then leads to vesicle-containing endosomes called multivesicular bodies (MVBs). These MVBs will then fuse with the plasma membrane, releasing their internal vesicles (the exosomes) into the extracelluar medium (Cvjetkovic et al. The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles. Journal of Extracellular Vesicles, 3: 23111.2014).
Exosomes are secreted by most cell types including epithelial cells, hematopoietic cells, dendritic cells, B cells, T cells, mast cells, platelets, microglia and some tumor cells. Exosomes can be found in various body fluids including urine, saliva, plasma, serum, amniotic fluid, bronchoalveolar fluid and breast milk.
Increasing evidence has suggested that exosomes play an important role in cell-to-cell signaling. In particular, exosomes have been shown to contain cell-specific proteins, lipids and RNAs, which are transported to other cells, where they can alter function and/or physiology. Depending on the cellular origin, exosomes may contain various cellular proteins including MHC molecules, tetraspanins, adhesion molecules and metalloproteinases. In addition to the exosomal proteins, mRNA and miRNA has been recently reported to be found in exosomes, which has brought the attention of many researchers to explore the role of exosomes. Moreover, it has been shown that these exosomal mRNAs can be translated into proteins by recipient cells and that the exosomal miRNAs are able to modulate gene expression in recipient cells.
Exosomes have been shown to be involved in the pathogenesis of cancer and degenerative diseases. Therefore, analysis of exosomal contents can be potentially used for non-invasive diagnostics of cancer and other disorders.
There are a number of different methods for isolating exosomes. The original and most commonly used method involves multiple centrifugation and ultracentrifugation steps (see Thery et al. Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids. Unit 3.22, Subcellular Fractionation and Isolation of Organelles, in Current Protocols in Cell Biology, John Wiley and Sons Inc., 2006). There are numerous drawbacks associated with the use of ultracentrifugation for exosome isolation. First, this method is not specific for exosomes and will co-purify larger vesicles, protein aggregates and even ribosomes. In addition, this method is time consuming and labour intensive, as it can involve as many as 5 centrifugation steps, with some of the steps requiring speeds of 100,000×g for several hours. Furthermore, this method requires the use of expensive and specialized ultracentrifuges.
Newer methods have been described, which are based upon immuno-magnetic capture of exosomes using magnetic beads coated with antibodies directed against proteins exposed on exosomal membranes. While these antibody based methods eliminate the need for ultracentrifugation, these methods are not suitable for the purification of large amounts of exosomes and are still quite costly.
Methods based on the use of volume-excluding polymers, such as PEG, have been recently described by a number of different groups (U.S. Pat. Appl. 20130273544, U.S. Pat. Appl. 20130337440). Two such products are ExoQuick (System Biosciences, Mountain View, USA) and Total Exosome Isolation Reagent (Life Technologies, Carlsbad, USA). These polymers work by tying up water molecules and forcing less-soluble components such as extracellular vesicles, as well as proteins out of solution, allowing them to be collected by a short, low-speed centrifugation. While the use of precipitation agents eliminate the need for ultracentrifugation and are less expensive that antibodies and beads, there is still the problem of contamination of the exosomes with protein aggregates and macromolecular complexes.